Enhances lak cell activation by treatment of human peripheral blood mononuclear cells with amino acid amides

Abstract

There is provided an improved process for enhanced LAK cell activation wherein the peripheral blood mononuclear cells are treated with an amino acid amide to yield depletion, prior to the lymphocytes being cultured at high density. Also provided are pharmaceutical compositions and methods of using them in combination with IL-2 to treat cancer in a mammal.

We claim: 
     
       1. In a process for preparing lymphokine-activated killer cells wherein peripheral blood mononuclear cells are cultured to produce a population of cells which when activated with a lymphokine are cytotoxic for natural killer cell-resistant tumor cells, the improvement comprising contacting said peripheral blood mononuclear cells or peripheral blood lymphocytes resulting therefrom with an L-amino acid amide, wherein the L-amino acid is selected for the group consisting of leucine, isoleucine, phenylalanine, and valine, or a mixture of any of the foregoing, and thereafter culturing the resulting cells. 
     
     
       2. A process according to claim 1 wherein said peripheral blood mononuclear cells are human cells. 
     
     
       3. A process according to claim 2 wherein the contacting is performed for a period of about 40 minutes. 
     
     
       4. A process according to claim 3 wherein the amino acid amide is phenylalanine. 
     
     
       5. A process according to claim 4 wherein the phenylalanine amide is present in a concentration of about 1 to 10 mM. 
     
     
       6. A process according to claim 5 wherein the phenylalanine amide is present in a concentration of about 10 mM. 
     
     
       7. A process according to claim 3 wherein the human peripheral blood mononuclear cells or peripheral blood lymphocytes resulting therefrom are cultured in the presence of interleukin-2. 
     
     
       8. A process according to claim 5 wherein the human peripheral blood mononuclear cells or peripheral blood lymphocytes resulting therefrom are cultured in the presence of interleukin-2. 
     
     
       9. A process according to claim 7 wherein the human peripheral blood lymphocytes obtained by contacting with the amino acid amide are washed and resuspended. 
     
     
       10. A process according to claim 8 wherein the human peripheral blood lymphocytes obtained by contacting with phenylalanine amide are washed and resuspended. 
     
     
       11. A process according to claim 9 wherein the resuspended human peripheral blood lymphocytes are cultured for 2 to 4 days in the presence of recombinant interleukin-2. 
     
     
       12. A process according to claim 10 wherein the resuspended human peripheral blood lymphocytes are cultured for 2 to 4 days in the presence of recombinant interleukin-2. 
     
     
       13. A process according to claim 11 wherein the concentration of human peripheral blood lymphocytes is from about 3×10 6  cells/mL to about 1×10 8  cells/mL. 
     
     
       14. A process according to claim 12 wherein the concentration of human peripheral blood lymphocytes is from about 3×10 6  cells/mL to about 1×10 8  cells/mL. 
     
     
       15. In a process for preparing lymphokine-activated killer cells wherein peripheral blood mononuclear cells are cultured to produce a population of cells which when activated with a lymphokine are cytotoxic for natural killer cell-resistant tumor cells, the improvement comprising contacting said peripheral blood mononuclear cells or peripheral blood lymphocytes resulting therefrom with a combination of an L-amino acid amide and an L-amino acid lower alkyl ester, wherein the L-amino acid amide is selected from the group consisting of leucine, isoleucine, phenylalanine, and valine, and the L-amino acid lower alkyl ester is selected from the group consisting of alanine, aspartic acid, cysteine, glutamic acid, glutamine, phenylalanine, proline, tyrosine, tryptophan, and valine, and thereafter culturing the resulting cells. 
     
     
       16. A process according to claim 15 wherein the peripheral blood mononuclear cells are human. 
     
     
       17. A process according to claim 16 wherein the contacting is performed for a period of about 20 to 40 minutes. 
     
     
       18. A process according to claim 17 wherein the L-amino acid amide is phenylalanine. 
     
     
       19. A process according to claim 18 wherein the ester is L-phenylalanine methyl ester and the amide is phenylalanine amide. 
     
     
       20. A process according to claim 19 wherein the ester is present in a concentration of about 1-5 mM and the amide is present in a concentration of about 1-10 mM. 
     
     
       21. A process according to claim 20 wherein the hydrogen chloride salt of the ester and the amide is present. 
     
     
       22. A process according to claim 19 wherein the human peripheral blood mononuclear cells or peripheral blood lymphocytes are cultured in the presence of interleukin-2. 
     
     
       23. A process according to claim 20 wherein the human peripheral blood mononuclear cells or peripheral blood lymphocytes are cultured in the presence of interleukin-2. 
     
     
       24. A process according to claim 22 wherein the peripheral blood lymphocytes obtained by contacting with the ester and amide are washed and resuspended. 
     
     
       25. A process according to claim 23 wherein the peripheral blood lymphocytes obtained by contacting the ester and amide are washed and resuspended. 
     
     
       26. A process according to claim 24 wherein the resuspended peripheral blood lymphocytes are cultured for 2-4 days in the presence of recombinant interleukin-2. 
     
     
       27. A process according to claim 25 wherein the resuspended peripheral blood lymphocites are cultured for 2-4 days in the presence of recombinant interleukin-2. 
     
     
       28. A process according to claim 26 wherein the concentration of peripheral blood lymphocytes is from about 3×10 6  cells/mL to about 1×10 8  cells/mL. 
     
     
       29. A process according to claim 27 wherein the concentration of peripheral blood lymphocytes is from about 3×10 6  cells/mL to about 1×10 8  cells/mL.